Fig 1: HoxC5 represses hTERT expression through its binding to hTERT upstream enhancer region. a ChIP-Seq results indicate the binding of HoxC5 and Pbx4 at −20 kb upstream region (highlighted by yellow) of hTERT TSS (Highlighted by green) that show enrichment of H3K27ac and H3K4me1 specifically in telomerase-positive embryonic stem cells (WA01) and cancer cells (PC-3), but not in telomerase-negative primary fibroblast cells (IMR90). The interaction between hTERT promoter and the −20 kb enhancer region can be observed from 4C data done in A375 and BLM cells. Cis-interactions with FDR < 0.05 were selected for visualization (arcs). The blue arcs represent the interaction of hTERT promoter with HoxC5 and Pbx4-binding region (FDR = 6.3E-137). The gray color arcs are other significant interactions with hTERT promoter. b ChIP was performed against RNA polymerase II (Pol2), H3K4me1, and H3K27ac in PC-3 cells overexpressing control GFP or Flag-tagged HOXC5 followed by qPCR with primers specific for hTERT TSS, −10 kb and −20 kb upstream enhancer regions as indicated. c ChIP was performed against RNA polymerase II (Pol2), H3K4me1, and H3K27ac in A375 cells overexpressing control GFP or Flag-tagged HOXC5 followed by qPCR with primers specific for hTERT TSS, −10 kb and −20 kb upstream enhancer regions as indicated. d ChIP was performed against RNA polymerase II (Pol2), H3K4me1, and H3K27ac in WA01 cells before (P0) and after neural differentiation (P4) followed by qPCR with primers specific for hTERT TSS, −10 kb and −20 kb upstream enhancer regions as indicated. e The expression of endogenous hTERT mRNA in A375 cells co-expressing dCas9-KRAB with control sgRNAs or sgRNAs targeting the HoxC5-binding region at −20 kb upstream hTERT enhancer. f, The top ten biological processes associated with endogenous HoxC5-binding sites in PC-3 cells. The p value is based on the Binomial or Hypergeometric test. The pathways involved in cell differentiation are highlighted in red. vp view point
Fig 2: HOXC5 suppresses hTERT expression and inhibits cancer cell growth. Representative images (a) and quantification (b) of in vitro colony formation assay in PC-3 cells expressing GFP, mir-615-3p, HOXC5 or both HOXC5 and mir-615-3p. Tumor growth curve (c) representing images (d), and tumor weight (e) showed growth of xenograft tumors in NSG mice injected with PC-3 cells expressing GFP, mir-615-3p, HOXC5 or both HOXC5 and mir-615-3p in vivo. Significance was determined by t test. *P < 0.05. Scatterplots showing Spearman correlation between hTERT and HOXC5 (f, i); miR-615-3p and hTERT (g, j); HOXC5 and miR-615-3p (h, k) in THYM (top) and TGCT (bottom) cancer samples. Red line indicates the linear fit with 95% confidence interval in shaded gray. All values are in RSEM log2 units. TGCT testicular germ cell tumor; THYM thymoma
Fig 3: HoxC5 inhibits hTERT expression. a A schematic representation of mir-615-3p genomic localization in the intron of HOXC5. b, c Relative hTERT mRNA expression and telomerase activity in HeLa, PC-3, U251, or BT549 cells transduced with lentivirus overexpressing Flag-tagged HOXC5. The telomerase activity in cells transiently transfected with control GFP vector is set as (1). d Telomere length in HeLa, PC-3, U251, or BT549 cells transduced with lentivirus overexpressing GFP or Flag-tagged HOXC5. PD: population doubling. e The expression of endogenous HOXC5 mRNA in HeLa cells expressing control shRNA or two independent anti-HOXC5 shRNAs. f Relative hTERT mRNA expression in HeLa cells expressing control shRNA or two independent anti-HOXC5 shRNAs. g Telomere length in HeLa cells transduced with lentivirus overexpressing control shRNA or two independent anti-HOXC5 shRNAs. Significance was determined by t test. *P < 0.05. PD population doubling
Fig 4: HoxC5 recruits Pbx4 and Meis3 to suppress hTERT expression in HeLa cells. a Schematic representation of HOXC5 function domains with its HX and HD motifs highlighted in black. The mutations introduced in HX or HD motif are illustrated, respectively. b, c Relative hTERT mRNA expression and telomerase activity in HeLa cells transduced with lentivirus overexpressing Flag-tagged GFP, wild-type (WT), or mutants (M1 or M2) HOXC5. d Western blotting shows the expression of Flag-tagged GFP, wild-type (WT) and mutants (M1 or M2) HoxC5 expressed in HeLa cells. e Immunoprecipitation of V5-tagged HoxC5 resulted in specific co-immunoprecipitation of Flag-tagged Pbx4, but not Flag-tagged Pbx1, 2 or 3 in HeLa cells. f The purified recombinant 6xHis-tagged HoxC5 proteins from bacteria interact with the in vitro translated and 35S-Methionine labeled Pbx4 proteins, but not Pbx1, 2, or 3. g Immunoprecipitation of Flag-tagged HoxC5 resulted in co-immunoprecipitation of V5-tagged Meis3, but not V5-tagged Meis1 or 2 in HeLa cells. h, Transient transfection of plasmids co-expressing PBX4 or MEIS3 with HOXC5 results in synergistic suppression of hTERT mRNA expression in HeLa cells. Significance was determined by t test. *P < 0.05
Fig 5: Expression of hTERT, miR-615-3p, HOXC5, PBX1–4, and MEIS1–3 in WA01 human ES cells upon neural induction. a A schematic representation of the human ES cells neural differentiation process in monolayer culture. The details are described under “Methods”. b The expression of pluripotency genes (NANOG and OCT-4) and neurodevelopmental gene PAX6 during neural differentiation (passage 0–4) in WA01 embryonic stem cells were quantified by real-time RT-PCR. The expression of hTERT mRNA. c miR-615-3p (d) and HOXC5 mRNA (e), during neural differentiation (passage 0–4) in WA01 cells was quantified by real-time RT-PCR as indicated. f The expression of PBX1–4 and MEIS1–3 in P0 and P3 of WA01 neural differentiation was quantified by real-time RT-PCR as indicated
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